Figure 3.
Trogocytosis strips Her2 from SKOV3 target cells. (A) Her2 CAR GFP or control GFP macrophages (GFP; green) were incubated with SKOV3 cells for 2 h. The cells were then stained for Her2 (magenta) and DAPI (blue). Her2 CAR macrophages showed bright puncta of internalized Her2 (yellow arrowhead). (B) For quantitative analysis of Her2 surface levels, Her2 CAR GFP or control GFP macrophages were incubated with SKOV3 cells (mCh-CAAX) for 2 h. The cells were then stained for Her2 and analyzed by flow cytometry. A representative flow histogram shows the Her2 levels on SKOV3 cells alone (pink) and SKOV3 cells incubated with Her2 CAR (blue) or control GFP macrophages (green). SKOV3 cells were also stained with an isotype control antibody as a negative control (gray). The graph depicts median fluorescence intensity of Her2. (C) Removal of a generic membrane tethered protein, mCh-CAAX, was quantified on the same SKOV3 cells. The graph depicts median fluorescence intensity of mCh-CAAX. (D) MHC-1 (HLA A–C) and CD47 removal was measured by antibody staining followed by flow cytometry. The percent remaining signal for Her2, MHC-1, and CD47 was calculated by dividing the signal in SKOV3 cells incubated with Her2 CAR macrophages by the signal in SKOV3 alone. Graphs B–D show data from N = 4 independent replicates. Bars represent the mean ± SEM of the replicates. Data were compared using a one way ANOVA with a Holm–Sidak multiple comparison test. Data collected on the same day are annotated with the same shape point. ** denotes P < 0.005; **** denotes P < 0.0005. Scale bar denotes 20 μm. Refer to the image caption for details.

Trogocytosis strips Her2 from SKOV3 target cells. (A) Her2 CAR GFP or control GFP macrophages (GFP; green) were incubated with SKOV3 cells for 2 h. The cells were then stained for Her2 (magenta) and DAPI (blue). Her2 CAR macrophages showed bright puncta of internalized Her2 (yellow arrowhead). (B) For quantitative analysis of Her2 surface levels, Her2 CAR GFP or control GFP macrophages were incubated with SKOV3 cells (mCh-CAAX) for 2 h. The cells were then stained for Her2 and analyzed by flow cytometry. A representative flow histogram shows the Her2 levels on SKOV3 cells alone (pink) and SKOV3 cells incubated with Her2 CAR (blue) or control GFP macrophages (green). SKOV3 cells were also stained with an isotype control antibody as a negative control (gray). The graph depicts median fluorescence intensity of Her2. (C) Removal of a generic membrane tethered protein, mCh-CAAX, was quantified on the same SKOV3 cells. The graph depicts median fluorescence intensity of mCh-CAAX. (D) MHC-1 (HLA A–C) and CD47 removal was measured by antibody staining followed by flow cytometry. The percent remaining signal for Her2, MHC-1, and CD47 was calculated by dividing the signal in SKOV3 cells incubated with Her2 CAR macrophages by the signal in SKOV3 alone. Graphs B–D show data from N = 4 independent replicates. Bars represent the mean ± SEM of the replicates. Data were compared using a one way ANOVA with a Holm–Sidak multiple comparison test. Data collected on the same day are annotated with the same shape point. ** denotes P < 0.005; **** denotes P < 0.0005. Scale bar denotes 20 μm.

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